Method and device for growing and/or treating cells

ABSTRACT

The invention relates to a method for growing and treating cells in an automated manner, for diagnostic purposes. The inventive method involves a cell culture plate comprising a plurality of bore holes for receiving cells, the holes being open on the upper side of the cell culture plate and closed on the lower side of the same. Oxygen and nutrients are dynamically supplied to the bore holes used as cell culture chambers.

FIELD OF THE INVENTION

The invention relates to a method for growing and/or treating cells inan automated manner for diagnostic purposes. The invention furthermorealso relates to a device for growing and/or treating cells and the useof a cell culture plate for this purpose.

BACKGROUND OF THE INVENTION

In the earlier DE 199 35 643.2 A1, a device for growing and/or treatingcells is described, a moldable cell culture chamber being arranged on acarrier. The cell culture chamber is in this case formed by the carrieror a carrier film on one side and a cell culture film on the other side,which is elastic. Using a device of this type, it is possible to carryout a mass culture of cells with great variability and for many intendeduses.

For further prior art, reference is made to DE 197 19 751 A1.

SUMMARY OF THE INVENTION

The present invention is based on the object of creating a method and adevice for growing and/or treating cells in an automated manner, itbeing possible in a space which is as small as possible to grow and/ortreat a high number of cells, which can also be of different type, fordiagnostic purposes.

According to the invention, this object is achieved by the methodmentioned in claim 1.

In claims 5 and 6, in each case a device for carrying out the method isdescribed.

The use of a cell culture plate known per se to achieve the object setis shown in claim 13.

Cell culture plates of this type are known generally in medicine, inparticular in pharmaceutical research, under the well plate or multiwellplate make. Here, cells for analytical tests are introduced into boresor hollows of the cell culture plate. The cells are then brought intocontact with a very wide variety of substances by pipetting, and theiractions on the cells are observed.

According to the invention, however, in complete modification of theprevious use of a cell culture plate of this type, this is used forgrowing and/or treating cells in a dynamic method. This means, via thesupply of oxygen and/or nutrients, which takes place continuously oralternatively batchwise, cells can now be grown and/or treated andobserved over a longer period of time. The supply of nutrients andoxygen necessary for this can take place in a very varied way.

In a simple manner, nutrients and oxygen can here be led togetherthrough a continuous or alternatively batchwise perfusion of the hollowsor bores now converted to cell culture chambers.

In a very advantageous embodiment of the invention, it is possible,however, to introduce a separate supply of oxygen into the cell culturechamber from the underside of the cell culture plate through agas-permeable film or membrane. Normally, the cell culture plates areprovided with stable, gas-impermeable bottoms. If these bottoms are nowreplaced by an appropriate gas-permeable film or membrane and anappropriate oxygen or air supply is provided to these areas, a simpleand very intensive supply of oxygen is provided to the cells.

A further possibility for the supply of nutrients and/or oxygen canconsist in that, in at least some of the bores or cell culture chambersof the cell culture plate, inserts can be employed which in each caseare provided with supply bores and return flow bores.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described, by way of example, with referenceto the accompanying drawings in which:

FIG. 1 shows a cell culture plate (multiwell plate) in perspective view;

FIG. 2 shows a section according to the line II—II of FIG. 1 inmagnified representation with the construction of the bores of the cellculture plate, in each case as a cell culture chamber;

FIG. 3 shows a section based on the line III—III of FIG. 2;

FIG. 4 shows an alternative embodiment of the construction of cellculture chambers in a section similar to that based on FIG. 2;

FIG. 5 shows a further embodiment for the formation of cell culturechambers, likewise in a section corresponding to that based on FIG. 2;

FIG. 6 in principle shows the supply to and removal of nutrients fromthe cell culture chambers in a plan view; and

FIG. 7 shows, sector-wise, an embodiment where the cell culture chamberscan be placed under pressure.

DETAILED DESCRIPTION OF THE INVENTION

For growing and/or treating cells for diagnostic purposes, a cellculture plate 1 (multiwell plate) known per se is used, which in generalhas a high number of holes or bores 2 according to its size. The bores 2have diameters of a few millimeters up to a number of millimeters, andtheir number can be up to several hundred units per cell culture plate1.

From FIGS. 2 to 5, various embodiments of the bores 2 and theirconversion to give cell culture chambers 2′ are apparent. According toFIG. 2, in each bore 2 which is to serve as a cell culture chamber 2′ aninsert 3 is employed which is provided with a supply bore 4 and a returnflow bore 5, which extend from the upper side of the cell culture plate1 down to its underside, the undersides of the cell culture plate or thebores 2 being closed by bottoms 6. The inserts 3 are thus inserted fromthe upper side of the cell culture plate 1 into the open bores 2. Forthe sealing of the cell culture chambers 2′, the inserts 3 are in eachcase provided on the peripheral side with a sealing ring 19.

From the side of an insert 3, in each case pointed to the bottom 6, aseparating bridge 7 projects in the direction of the bottom 6. Theseparating bridge 7 lies between the supply bore 4 and the return flowbore 5 and extends, as is apparent from FIG. 3, straight across the bore2. By means of the separating bridge 7, it is achieved that thenutrients introduced via the supply bore 4 cannot flow in a“short-circuit” directly to the return flow bore 3. By means of theseparating bridge 7, they are forced, correspondingly, to flow throughthe entire cell culture chamber 2′, and in the course of this to flowover the cells 8 applied to the bottom 6. At the same time, theseparating bridge 7 can also serve for the centering or for the easierintroduction of the insert 3 into the bore 2.

In the embodiment with the insert 3 shown in FIG. 2 on the left, thesupply of oxygen to the cells 8 takes place together with the supply ofnutrients via the supply bore 4. If the fixed bottom 6 is removed andthis is replaced by a gas-permeable film or membrane 9 corresponding tothe embodiment shown greatly enlarged in thickness on the right in thefigure, a very intensive and direct supply of oxygen from the undersideof the cell culture plate 1 through the gas-permeable film can takeplace (see arrows). The growth and the treatment of the cells 8 takesplace on the gas-permeable film 9, which is shown significantly thickerin the figures for reasons of clarity.

FIG. 4 shows, instead of a large number of inserts 3 to be inserted intothe bores 2, a cover plate 10, which is shown only very generally,marked with dashes, in FIG. 1. In the cover plate 10, supply connections4′ and return flow connections 5′ are inserted into corresponding boresin the cover plate. As is apparent, the supply connections 4′ projectmarkedly deeper into the accompanying bores 2 or the cell culturechambers 2′ in comparison with the return flow connections 5′. As isfurther apparent, by means of this measure it is likewise achieved thatno short-circuit flow takes place directly from a supply to a returnflow. The cells 8 are on the contrary flowed over by the nutrientmedium. In FIG. 4, the two variants having a fixed bottom 6 and agas-permeable film or membrane 9 are likewise shown next to one another.

On the left in FIG. 4, it is shown how the cover plate 10 is connectedto the cell culture plate 1 by screws 11. On the right, it is shownthat, instead of or additionally to a connection via screws 11, sealingby means of a sealing arrangement 12 between the cover plate 10 and thecell culture plate 1 can also be achieved.

In FIG. 5, in principle, a two-part construction of the cover plate 10with an upper cover 10 a and a lower cover 10 b is shown. As isapparent, the upper cover 10 a and the lower cover 10 b are connected toone another at an adjustable distance. By means of springs 13 placedbetween them, a pretension is achieved. The upper cover 10 a is attachedby means of bridges 14 to the edges of the cell culture plate 1 runninground these. The lower cover 10 b is provided with bores, through whichsupply connections 4′ and return flow connections 5′ are pushed,similarly to the embodiment according to FIG. 4. The supply connections4′ and return flow connections 5′ are correspondingly also to be led outthrough the upper cover 10 a either to its upper side or optionally alsoat the side on the edges. By means of the springs 13, a sealing of thecell culture chambers is achieved, because the lower cover 10 b liessuitably tightly on the upper side of the cell culture plate 1. At thesame time, extensions 15, which, on the underside of the lower cover 10b, project downward from this, are in this way also inserted into thebores 2 to form closed cell culture chambers 2′.

From FIG. 5, it is also apparent in the representation marked by dashesthat the entire unit containing the cell culture plate 1 can be placedin a tank 16. The cell culture plate 1 is in this case attached runninground tightly on the tank 16. Between the bottom formed in this casefrom a gas-permeable film or membrane 9 and the tank bottom is situatedan interspace 17. If oxygen is introduced into the tank 16, e.g. viabores 18, a very intensive direct supply of oxygen for the cells 8 lyingin the cell culture chambers 2′ takes place through the film or membrane9.

The bores 18 can preferably correspond to the size of the bores 2. Theinterspace 17 can optionally be inappropriate, in this case the cellculture plate 1 attaching directly to the bottom of the tank 16 and itbeing possible for an exchange of gas to take place via an opening,preferably corresponding to the diameter of the cell culture chambers2′, by means of a gas-permeable film 9 or membrane 9. The bores 18 inthe tank 16 additionally make possible a visual improvement onmicroscopic observation of the cells. Preferably, for this the materialof the cover plate 10 and also of the tank 16 is also produced fromtransparent material, such as, for example, polycarbonate orpolystyrene.

Using the method and the device according to the invention, a type ofbioreactor for growing and/or treating cells is [lacuna] from a cellculture plate 1 known per se. By means of the dynamic supply of nutrientmedium and oxygen, a long-term culture is also possible here.

A further advantage of the invention is the possibility of automation ofroutinely proceeding processes, such as, for example, of any desiredupwardly open culture vessels.

Cell culture plates of conventional construction known per se can alsobe modified for the method according to the invention in a simplemanner. Thus it is possible, for example, to remove the fixed bottom 8in a simple manner and to stretch a gas-permeable film 9 over the bores2, which are now open on both sides, on the underside of the cellculture plate 1. By means of the gas-permeable film 9, the developmentof the cells 8 can also be observed if necessary, for which purpose thefilm 9 is to be designed to be suitably transparent.

In order that the cells 8 grow or are arranged in a very defined manneras a layer having a two-dimensional spread in the cell culture chambers2′, the entire cell culture plate 1 can, if necessary, also be frozen ifit is wanted to transport or to store them intermediately until theiruse.

As a rule, all or a major part of the bores 2 of a cell culture platewill be converted to cell culture chambers 2′ for the culture method. Ifnecessary, it is, of course, also possible also to create onlyindividual cell culture chambers 2′ having correspondingly individualinserts 3, since the inserts 3 can also be employed on their own. Theinserts 3 can also be flowed toward or emptied individually in separateculture units.

It is significant in each case that care is taken for an adequate supplyof oxygen to the cells 8, in order also to be able to carry out alonger-lasting cell culture method, in particular for “demanding” cells.On the basis of the embodiment of the known cell culture plates 1, inparticular of the bores 2, growth and/or treatment of cells in the formaccording to the invention was hitherto not possible.

In FIG. 6, in principle the supply to and the removal of nutrients andoptionally of oxygen from the cell culture chambers 2 is shown in a planview. As is apparent, a common supply connection 20 is provided here,from which via a number of branch lines 21 the individual cell culturechambers 2 are supplied with nutrient medium and optionally also withoxygen. A joint removal via an outlet connection 23 then in turn followsvia individual branch lines 22.

By means of the common supply connection 20 and the common outletconnection 23, the device can be used, for example, in the form of a“docking station”, which is connected to a central station, possiblytogether with other devices. In this way, very large breeding stocksand/or growth devices can be created.

The joint supply and the joint removal using the branch lines 21 and 22can take place, for example, through appropriate lines, openings orcannulas in the cover plate 10, from where the cell culture chambers 2can in each case then be supplied separately via individual branch lines21. In combination with the tank 16, in this way a type of cassettestructure can be created. If necessary, a number of units can also bearranged one above the other. This is possible, for example, in that anumber of cell culture plates 1 are arranged one above the other, itbeing possible to carry out the supply with nutrient medium and withoxygen through cover plates 10 lying in between, which arecorrespondingly provided, e.g. in each case on their upper side and ontheir underside, with common supply connections 20, branch lines 21 and22 and common outlet connections 23.

Depending on the size of the cell culture chambers 2 and their number,it may optionally be necessary that a number of cover plates 10 withsupply connections 20 arranged therein, branch lines 21 and 22 andoutlet connections 23 are provided, which for reasons of space areprovided with branch lines 21 and 22 arranged suitably displaced, inorder that all cell culture chambers 2 can be supplied.

Additionally and alternatively, the cell culture plates 1 themselves canalso be provided in each case with common supply connections 20, branchlines 21 and 22 and with common outlet connections 23, in order to beable to reach each cell culture chamber 2. For this, it is possible, forexample, to divide a cell culture plate 1 in the middle and to introducethe flow channels in the area of the plane of separation. In each case,however, a channel guide should be created, by means of which a uniformsupply of all cell culture chambers 2 is guaranteed.

The film or the membrane 9 can, if necessary, also be of microporousdesign, which has the advantage that nutrient medium can be brought intothe cell culture chambers 2′ not only from above, but additionally oralternatively also from below.

In FIG. 7, a very advantageous embodiment is shown, it being possiblefor the cell culture chambers 2′ to be placed under pressure. For this,the cell culture plate 1 is closed off on the upper side by apressure-tight dome 24 and on the underside by a pressure-tightcontainer 25. The openings 2 on the upper side of the cell culture plate1 are closed off using a flexible film 26, which if necessary can alsobe gas-permeable or microporous in order to achieve from this side asupply of the cells 8 situated in the cell culture chambers 2′ withoxygen/air and/or nutrient medium. On the underside, the cell culturechambers 2′ are likewise closed off by the membrane 9, which is ofgas-permeable or microporous design.

By means of pressure connections 27 which are not shown in greaterdetail, a chamber 28 between the dome 24 and the film 26 and a chamber29 between the membrane 9 and the bottom of the container 25 can beplaced under elevated pressure or reduced pressure by means ofappropriate pressure sources. The control of the pressure conditions isarbitrary here. This means pressure can be exerted alternately on thecell culture chambers 2′ from above or from below or alternatively atthe same time. For this, the film 26 and the membrane 9 are deformedcorrespondingly (see dashed representation). In this way, the cells 8are accordingly moved mechanically, which has, for example, considerableadvantages for the production of tissues, such as, for example, bone,cartilage, musculature and the like, since owing to extensions in thesense of conditionings or [lacuna], training can be simulated.Additionally, rhythmical or intermittent pressure loadings can beproduced in the cell culture chambers 2′. By means of these measures, invivo conditions can be simulated better.

By the use of the film 26, the cell culture chambers 2′, if necessary,are also accessible to pipetting processes.

Instead of a simple film 26, the openings 2 can also be covered by aseptum-like plastic membrane.

Instead of pressure loadings or alternatively additionally, electricalcurrents can be imposed on the cells 8. In this way, for example,extensions can also be induced by means of electromagnetic fields, whichis advantageous, for example, for cardiac muscle cells, CNS cells(neuronal cells). In this way, for example, it is possible to testmedicaments which accelerate or reduce the heart rate, for which purposethe electrical currents in practice electrically stimulate cardiacmuscle cells. In this way, interactions occur between a technologicaland a biological system.

The cell culture systems described above can, if necessary, also becombined with one another in the form of a bioreactor or in a sandwichsystem. For this, it is only necessary to provide, instead of a tank 16or the container 25, an appropriately shaped intermediate bottom, sothat—as indicated marked by dashes in FIG. 7—a further unit 30 connectsunder the container 25 or under a corresponding intermediate bottom. Atthe same time, a number of units of this type can be arranged one abovethe other. The bioreactor resulting here can in this case be constructedin stage-like form or alternatively in mirror image form, a further unitconnecting in mirror image form to the unit described in FIG. 7. In thiscase, the space 29 serves for the supply of air/oxygen and/or nutrientmedium both for the cell culture plate 1 and for the cell culture plate(not shown) located in the unit 30. Of course, if necessary, however,separate supplies and outlets are also possible.

Instead of simple cells 8 in the cell culture chambers 2′, multilayercultures can, of course, also be grown.

One of the essential differences from the prior art to be retained isthat the present cell culture system concerns dynamic processes whichcan proceed continuously or intermittently and not only static growingof cell culture systems, which after starting are no longer additionallytreated.

Fundamentally, in the present system three methods with accompanyingdevices are present, namely:

1. In the simplest case closed bottoms are present and the supply of thecell culture chambers 2′ takes place from above through the openings orbores 2 using corresponding supply bores 4 and return flow bores 5.

2. An embodiment of the cell culture plates 1 having a gas-permeable ormicroporous membrane 9 on the underside and closed-off bores 2 on theupper side, with or without supply bores 4 and return flow bores 5.

3. Elastic films both on the upper side and on the underside of the cellculture plate 1, which can be gas-permeable or microporous and which areloaded with appropriate pressures. The gas-permeable or microporousmembranes 9 on the underside can be formed from a large number ofindividual membranes which in each case cover the cell culture chambers2′ on the underside. In general, however, a film is used whichappropriately covers the entire underside of the cell culture plate 1,as is shown in FIG. 7.

According to the invention, complex cell culture systems, which placecorrespondingly higher demands on the microenvironment, can now betreated. At the same time, in the bores 2 of the cell culture plate 1, a3-D structure having two-dimensional spread can also be defined, whichcorresponds in vivo to the separation of a capillary from the nextcapillary. This bioartificial tissue section serves for the productionof organo-typical culture conditions in a very small space and thusmakes possible even more complex coculture systems using various celltypes and extracellular matrix to make high-throughput screeningaccessible.

Fundamentally, a thin-layer culture system is present in the methodaccording to the invention, which is oxygenated from below in anadvantageous way and which corresponds to the physiological celldensity, namely in the distance of a blood capillary in the organism tothe next blood capillary.

One of the main advantages of the present invention lies in theminiaturization with units which are as small as possible in a tightspace. In this way, a small number of cells is also needed for a culturemethod, which can then be appropriately proliferated during the method.

One of the main areas of use of the method according to the inventionand the device for this is therefore the investigation or the action ofchemicals and pharmaceuticals on cells, in particular on human cells. Inthis way, animal experiments which are very complicated and expensive tocarry out can be replaced at least partially.

1. A method for growing and/or treating cells for diagnostic purposesusing a cell culture plate (1), which has a large number cell culturechambers (2′), open on an upper side of the cell culture plate (1) andclosed on an underside thereof, in which cells are contained, havingsupply of oxygen and a flow of nutrients to cell culture chambers (2′),which are flowed through with at least the nutrients via supply bores(4) and return flow bores (5).
 2. The method as claimed in claim 1,wherein the nutrients flow through the cell culture chambers (2′) withor without oxygen.
 3. The method according to claim 1, wherein theoxygen is introduced into the cell culture chambers (2′) through agas-permeable design of the bottom (6).
 4. The method according to claim1, wherein the bottoms (6) or a membrane (9) provided instead of thebottoms (6) are microporous.
 5. The method according to claim 1, whereinthe cell culture chambers (2′) are pressurized with different pressures.6. A device for growing and/or treating cells for diagnostic purposescomprising a cell culture plate (1), which has a large number of cellculture chambers (2′) open on an upper side of the cell culture plate(1) and closed on an underside thereof, in which cells are contained,having a supply of oxygen and a flow of nutrients to the cell culturechambers (2′), which are flowed through with at least the nutrients viasupply bores (4) and return flow bores (5), and wherein at least some ofthe cell culture chambers (2) are closed on their undersides usinggas-permeable or microporous films or membranes (9) as bottoms (6).
 7. Adevice as claimed in claim 6, wherein in at least some of the cellculture chambers (2′) of the cell culture plate (1) inserts (3) can beemployed, which are in each case provided with supply bores (4) andreturn flow bores (5).
 8. The device as claimed in claim 7, wherein theinserts (3) are in each case provided on their sides pointing to thebottom (6) with a separating bridge (7) between the supply bores (4) andthe return flow bores (5).
 9. The device according to claim 8, whereinsupply connections (4′) and return flow connections (5′) in each caseextend from the supply bores (4) and return flow bores (5) toward thebottoms (6), the supply connections (4′) and the return flow connections(5′) in each case projecting into the cell culture chambers (2′) to adifferent extent.
 10. The device according to claim 7, wherein theinserts (3) are in each case provided with a sealing ring (19) on theperipheral side.
 11. The device according to claim 6, wherein the cellculture plate (1) is covered with a cover plate (10).
 12. The deviceaccording to claim 11, wherein the cover plate (10) is connected to thecell culture plate (1) by screws (11) and/or the cell culture plate (1)is sealed by a sealing arrangement (12).
 13. The device according toclaim 11, wherein the cover plate (10) is constructed in two parts,having an upper cover (10 a) and a lower cover (10 b), the two covers(10 a, 10 b) being connected to one another elastically or movably toone another.
 14. The device according to claim 13, wherein the lowercover (10 b) is provided with extensions (15) which project into atleast some of the cell culture chambers (2′), and which are providedwith supply connections (4′) and return flow connections (5′).
 15. Thedevice according to claim 10, wherein the cover plate (10) are providedwith at least one supply connection (20), with branch channels (21, 22)and with at least one outlet connection (23) for the supply of the cellculture chambers (2) with nutrient medium and/or oxygen.
 16. The deviceaccording to claim 15, wherein a number of cover plates (10) areprovided for the supply of one or more cell culture plates (1).
 17. Thedevice according to claim 6, wherein the cell culture plate (1) isconstructed in two parts, at least one supply connection (20), branchchannels (21, 22) and at least one outlet connection (23) being providedin the separating plane.
 18. The device according to claim 6, whereinthe cell culture plate (1) is inserted into a tank (16).
 19. The deviceaccording to claim 18, wherein the tank (16) is provided with oxygensupply lines and oxygen openings (18) in its wall.
 20. The deviceaccording to claim 6, wherein the cell culture chambers (2′) can beplaced under variable pressure by means of pressure connections (27).21. The device according to claim 20, wherein additionally to theelastic membrane (9), on the underside of the cell culture plate (1) thecell culture chambers (2′) on the upper side of the cell culture plate(1) is covered with an elastic film (26), and in that interspaces (28,29) above and below the cell culture plate (1) can be placed underpressure.
 22. The device according to claim 6, wherein the cover plate(10) and/or the tank (26) or the container (25) are constructed asintermediate containers, and in that via the intermediate container oneor more units (30) are connectable to common or to separate connectionsfor the formation of a bioreactor.
 23. The cell culture plate (1)according to claim 6, wherein the cell culture plate (1) is introducedinto a tank (16) and is provided with a cover plate (10) and a tankbottom for automated perfusion.
 24. A cell culture plate (1) for growingand/or treating cells for diagnostic purposes comprising a large numberof cell culture chambers (2′) open on an upper side of the cell cultureplate (1), which are closed off on an underside thereof by bottoms (6),for growing and/or treating cells (8), which are introduced into thecell culture chambers (2′), in an automated manner for diagnosticpurposes, wherein at least some of the bottoms (6) of the cell cultureplate (1) are one of gas-permeable and microporous.
 25. The method forgrowing and/or treating cells for diagnostic purposes according to claim1, wherein the cell culture plate (1) is introduced into a tank (16) andis provided with a cover plate (10) and a tank bottom for automatedperfusion.
 26. The method for growing and/or treating cells fordiagnostic purposes according to claim 1, wherein the cell culture plate(1) is frozen for purposes of transporting or storage prior to growingcells.
 27. The method for growing and/or treating cells for diagnosticpurposes according to claim 1, wherein the contained cells are movedmechanically within the bores by alternating pressure in the bores. 28.The method for growing and/or treating cells for diagnostic purposesaccording to claim 1, wherein the contained cells are moved by imposingelectrical currents on the cells.
 29. A method for growing and/ortreating cells for diagnostic purposes using a cell culture plate (1),which has a large number of cell culture chambers (2′) open on an upperside of the cell culture plate (1) and closed on an underside thereof,in which cells are contained, the method comprising the steps ofsupplying oxygen and a flow of nutrients to the cell culture chambers(2′) and providing flow through of at least the nutrients via supplybores (4) and return flow bores (5), and introducing oxygen into thecell culture chambers (2′) through gas-permeable bottoms (6) of the cellculture chambers (2′).
 30. A method for growing and/or treating cellsfor diagnostic purposes using a cell culture plate (1), which has alarge number of cell culture chambers (2′) open on an upper side of thecell culture plate (1) and dosed on an underside thereof, in which cellsare contained, the method comprising the steps of supplying oxygen and aflow of nutrients to the cell culture chambers (2′) and providing flowthrough of at least the nutrients via supply bores (4) and return flowbores (5), and wherein the cell culture chambers (2′) are pressurizedwith different pressures.
 31. A method for growing and/or treating cellsfor diagnostic purposes using a cell culture plate (1), which has alarge number of cell culture chambers (2′) open on an upper side of thecell culture plate (1) and closed on an underside thereof, in whichcells are contained, the method comprising the steps of supplying oxygenand a flow of nutrients to the cell culture chambers (2′) and providingflow through of at least the nutrients via supply bores (4) and returnflow bores (5), and wherein the cell culture plate (1) is introducedinto a tank (16) and is provided with a cover plate (10) and a tankbottom for automated perfusion.